Negative feedback regulation of FGF signaling levels by Pyst1/MKP3 in chick embryos

BACKGROUND: The importance of endogenous antagonists in intracellular signal transduction pathways is becoming increasingly recognized. There is evidence in cultured mammalian cells that Pyst1/MKP3, a dual specificity protein phosphatase, specifically binds to and inactivates ERK1/2 mitogen-activated protein kinases (MAPKs). High-level Pyst1/Mkp3 expression has recently been found at many sites of known FGF signaling in mouse embryos, but the significance of this association and its function are not known. RESULTS: We have cloned chicken Pyst1/Mkp3 and show that high-level expression in neural plate correlates with active MAPK. We show that FGF signaling regulates Pyst1 expression in developing neural plate and limb bud by ablating and/or transplanting tissue sources of FGFs and by applying FGF protein or a specific FGFR inhibitor (SU5402). We further show by applying a specific MAP kinase kinase inhibitor (PD184352) that Pyst1 expression is regulated via the MAPK cascade. Overexpression of Pyst1 in chick embryos reduces levels of activated MAPK in neural plate and alters its morphology and retards limb bud outgrowth. CONCLUSIONS: Pyst1 is an inducible antagonist of FGF signaling in embryos and acts in a negative feedback loop to regulate the activity of MAPK. Our results demonstrate both the importance of MAPK signaling in neural induction and limb bud outgrowth and the critical role played by dual specificity MAP kinase phosphatases in regulating developmental outcomes in vertebrates.     

A PATO-compliant zebrafish screening database (MODB): management of morpholino knockdown screen information

Background  

The zebrafish is a powerful model vertebrate amenable to high throughput in vivo genetic analyses. Examples include reverse genetic screens using morpholino knockdown, expression-based screening using enhancer trapping and forward genetic screening using transposon insertional mutagenesis. We have created a database to facilitate web-based distribution of data from such genetic studies.     

Standardization of a broth microdilution susceptibility testing method to determine minimum inhibitory concentrations of aquatic bacteria

A multiple laboratory study was conducted in accordance with the standards established by the Clinical and Laboratory Standards Institute (CLSI), formerly the National Committee for Clinical Laboratory Standards (NCCLS), for the development of quality control (QC) ranges using dilution antimicrobial susceptibility testing methods for bacterial isolates from aquatic animal species. QC ranges were established for Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658 when testing at 22, 28 and 35 degrees C (E. coli only) for 10 different antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, ormetoprim/sulfadimethoxine, oxolinic acid, oxytetracycline and trimethoprim/sulfamethoxazole). Minimum inhibitory concentration (MIC) QC ranges were determined using dry- and frozen-form 96-well plates and cation-adjusted Mueller-Hinton broth. These QC ranges were accepted by the CLSI/NCCLS Subcommittee on Veterinary Antimicrobial Susceptibility Testing in January 2004. This broth microdilution testing method represents the first standardized method for determining MICs of bacterial isolates whose preferred growth temperatures are below 35 degrees C. Methods and QC ranges defined in this study will enable aquatic animal disease researchers to reliably compare quantitative susceptibility testing data between laboratories, and will be used to ensure both precision and inter-laboratory harmonization.     

Loss of phylogenetic information in chorion gene families of Bombyx mori gene conversion

The silkmoth chorion has provided a stimulating model for the study of evolution and developmental regulation of gene families. Previous attempts at inferring relationships among chorion sequences have been based on pairwise comparisons of overall similarity, a potentially problematic approach. To remedy this, we identified the alignable regions of low sequence variability and then analyzed this restricted database by parsimony and neighbor-joining methods. At the deepest level, the chorion sequence tree is split into two branches, called "alpha" and "beta." Within each branch, early- and late-expressing genes each constitute monophyletic groups, while the situation with middle-expressing genes remains uncertain. The HcB gene family appears to be the most basal beta-branch group, but this conclusion is qualified because the effect of gene conversion on branching order is unknown. Previous studies by Eickbush and colleagues have strongly suggested that ErA, HcA, and HcB families undergo gene conversion within a gene family, whereas the ErB family does not. The occurrence of conversion correlates with a particular tree structure; namely, branch lengths are much greater at the base of the family than at higher internodes and terminal branches. These observations raise the possibility that chorion gene families are defined by gene conversion events (reticulate evolution) rather than by descent with modification (synapomorphy).      

Sequences sufficient for correct regulation of Sgs-3 lie close to or within the gene.

The Drosophila melanogaster 68C chromosomal locus is the site of a prominent polytene chromosome puff that harbors the genes Sgs-3, Sgs-7 and Sgs-8. These genes code for proteins that are part of the salivary glue that Drosophila larvae secrete as a means of fixing themselves to an external substrate for the duration of the pre-pupal and pupal period. The 68C glue genes are regulated by the steroid hormone ecdysterone, with the hormone required for both initiation and cessation of gene expression during the third larval instar. Previous work has defined sequences sufficient for expression of abundant levels of Sgs-3 mRNA at the correct time and in the correct tissue. We show here that sequences sufficient for normal tissue- and stage-specific accumulation of Sgs-3 RNA, but adequate only for low levels of expression, lie within 130 bp of the 5' end of the gene, or within the gene.     

Activation and inactivation of the GABAA receptor: insights from comparison of native and recombinant subunit assemblies.

gamma-Aminobutyric acid (GABA) acts as an inhibitory transmitter in the vertebrate central nervous system, often by interacting with the GABAA type of receptor. Molecular cloning techniques have shown that GABAA receptors are assembled from at least five types of subunit, some of which are present in multiple forms. In this review, the functional properties of native GABAA receptors are compared with those of recombinant receptors, created by expression of appropriate cDNAs in frog oocytes or transfected mammalian cells. Native receptors typically display multiple conductance levels in the open state, complex kinetics, a Hill slope greater than unity, voltage dependence, and desensitization at high agonist doses. All of these features can also be exhibited by recombinant receptors, but marked qualitative and quantitative distinctions exist between receptors containing different combinations of subunits. It is argued that these functional differences are likely to be exploited in vivo by the expression of multiple, physiologically distinct GABAA receptors, distributed in an adaptive fashion throughout the nervous system.     

The effects of tidal currents on the rhythm of feeding and digestion in Pecten maximus L.

The orientation of individuals in two populations of Pecten maximus L., from the west coast of Ireland, shows that they have a marked preference to face directly into a tidal flow. In both localities examined there was a reversal of tides and the members of the populations were divided equally for flood and ebb tides. Twenty-four hour in situ studies of the animals were made and at all stages of the tide individuals facing either due east or west were observed. A cyclical feeding pattern imposed by the reversal of tidal flow is proposed.The pH of various parts of the digestive tract was investigated and showed a wide range of values. The most acid region was that of the stomach. The variations in pH of all of the regions of the gut examined throughout a 24-h period closely followed the pattern of the tidal cycle.Histological analysis of the stomach and digestive diverticula of representative samples of scallops taken at regular intervals over the 24-h periods clearly indicated a diphasic pattern of digestion within the tubules of the digestive diverticula. A close correlation between the phases of intracellular digestion, the pH variations in different regions of the gut, and the tidal cycle indicate distinct feeding cycles in Pecten. Those scallops facing into the ebb current show the same diphasic patterns as those individuals facing the flood current, but are 6 h out of phase. The cycle is considered as a duplication of a diphasic feeding pattern. The tubules themselves undergo a digestive process similar to that in Lasaea rubra (Montagu). The digestive cells phagocytose food material, begin intracellular digestion, and increase in size until they obscure the lumina. The dispersal of waste material and residual bodies is accomplished dramatically by a dehiscence of the tubule cells together with a loss of both digestive and crypt cells. New tubules are regenerated from the apices of those tubules breaking down. In any section of the diverticula tubules in two different conditions are found. The cycle of digestion takes 24 h and in order to facilitate feeding at each 12-h tidal cycle the tubules are equally divided into two phases with one 12 h behind the other.     

Microcomputer interfacing to mass flow type gas controllers for biotechnology applications

Summary An Apple IIe microcomputer with an Isaac analog-digital package has been interfaced to Tylan proportionating mass flow gas controllers to regulate three substrate gases which are introduced into an anaerobic bioreactor. Advantages of the system include: straightforward interfacing and programming, accurate monitoring and controlling, and advanced real time control capabilities.